SLIT2/ROBO1-signaling inhibits macropinocytosis by opposing cortical cytoskeletal remodeling

Vikrant K Bhosle,Dana J Philpott,Sajedabanu Patel,Guang-Ying Liu,Jane Y Wu,Sergio Grinstein,Yi-Wei Huang,Tapas Mukherjee,Lisa A Robinson,Bo Wen Pang,Michael Glogauer

doi:10.1038/s41467-020-17651-1

Abstract

Macropinocytosis is essential for myeloid cells to survey their environment and for growth of RAS-transformed cancer cells. Several growth factors and inflammatory stimuli are known to induce macropinocytosis, but its endogenous inhibitors have remained elusive. Stimulation of Roundabout receptors by Slit ligands inhibits directional migration of many cell types, including immune cells and cancer cells. We report that SLIT2 inhibits macropinocytosis in vitro and in vivo by inducing cytoskeletal changes in macrophages. In mice, SLIT2 attenuates the uptake of muramyl dipeptide, thereby preventing NOD2-dependent activation of NF-κB and consequent secretion of pro-inflammatory chemokine, CXCL1. Conversely, blocking the action of endogenous SLIT2 enhances CXCL1 secretion. SLIT2 also inhibits macropinocytosis in RAS-transformed cancer cells, thereby decreasing their survival in nutrient-deficient conditions which resemble tumor microenvironment. Our results identify SLIT2 as a physiological inhibitor of macropinocytosis and challenge the conventional notion that signals that enhance macropinocytosis negatively regulate cell migration, and vice versa.

Highlights

Macropinocytosis is essential for myeloid cells to survey their environment and for growth of RAS-transformed cancer cells
We first investigated which Slit-binding Robo receptors are expressed in macrophages and found that Robo[1], but not Robo[2], messenger RNA is expressed in the RAW264.7 macrophage cell line and in primary murine bone marrow-derived macrophages (BMDM) (Fig. 1a)
ROBO1 protein was detected in RAW264.7 cells as well as in primary macrophages derived from human peripheral blood mononuclear (MDM) cells (Supplementary Fig. 1a)

Summary

Introduction

Macropinocytosis is essential for myeloid cells to survey their environment and for growth of RAS-transformed cancer cells. Macrophages, and immature dendritic cells (iDCs) constitutively sample their extracellular surroundings via macropinocytosis, a phenomenon brought about by active plasma membrane ruffling induced by remodeling of the cortical cytoskeleton[3,4] These cells internalize bacterial pathogenassociated molecular patterns (PAMPs), such as muramyl dipeptide (MDP), via macropinocytosis, thereby initiating an immune response[5]. KRAS-transformed cancer cells use macropinocytosis to internalize large proteins, such as albumin, from their extracellular environment, to subsequently break them down into amino acids, which enter the cell metabolism to promote cancer cell survival[11] This might be relevant in the tumor core, which contains low concentrations of amino acids, including glutamine (Glut)[17]. Inhibition of macropinocytosis could be of therapeutic benefit in this context[11]

Methods

Results

Conclusion